Article Figures & Data
Figures
- Figure 1.
Deletion of TREM-1 decreases DEN-induced liver tumorigenesis. A, WT and Trem1−/− male mice were given a dose of DEN (25 mg/kg) at 15 days of age. Numbers of tumors (≥0.5 mm) and maximal tumor sizes (diameters) in livers of WT (n = 9) and Trem1−/− (n = 7) mice 8 months after DEN injection; ND, not detectable. Bars represent mean ± SD, statistical significance is indicated (***P < 0.001). One of 2 similar experiments is shown. B, numbers of tumors and maximal tumor sizes in livers of male WT (n = 7) and Trem1−/− (n = 10) mice 14 months after DEN injection. Bars represent mean ± SD, and statistical significance is indicated (**P < 0.01, *** P < 0.001). One of 2 independent experiments with similar results is shown. Representative microscopic pictures of livers from WT, Trem1−/− mice 8 months (C) and 14 months (D) after DEN injection. Arrowhead indicates tumor nodules. E, histologic analysis (hematoxylin and eosin) of livers from indicated mice 8 months (E) and 14 months (F) after injection of DEN; N, noncancerous liver tissues; T, tumors. Arrowheads indicate border between noncancerous liver tissues and tumor; original magnification, ×10. Bars, 50 μm. The expression of HCC marker α-fetoprotein (AFP) by liver tumor cells determined by immunohistochemistry (G) and by Western blot analysis (H) using an anti-AFP antibody; bar, 20 μm. One of 2 similar experiments is shown.
- Figure 2.
TREM-1 deficiency decreases of DEN-induced hepatic injury. WT and Trem1−/− male mice were treated for 4 hours with DEN (100 mg/kg) or left untreated. A and B, livers of WT or Trem1−/− mice were assessed for apoptosis by TUNEL staining. Original magnification, ×40. Values are mean ± SD for independent animals (n = 5). One of 4 similar experiments is shown. C, levels of IL-6 and ALT (F) in serum of WT and Trem1−/− mice treated with DEN at indicated time. D and E, hepatocyte proliferation in livers of WT or Trem1−/− mice was assessed by injecting mice with BrdUrd (1 mg/mL) 2 hours before the liver was removed. BrdUrd-positive cells were identified by immunostaining. Original magnification, ×40. Values are mean ± SD for independent animals (n = 3). One of 3 similar experiments is shown. Data are shown as mean ± SD (n = 5 mice per group). For all panels statistical significance is indicated (*P < 0.05, **P < 0.01); h, hour. One of 4 similar experiments is shown.
- Figure 3.
Loss of TREM-1 alters recruitment of neutrophils and monocytes. Bone marrow, blood, and liver samples of indicated mice were stained with appropriate mAbs and analyzed by flow cytometry. Data are shown as mean ± SD (n = 3 mice per group) of neutrophils (CD11bhigh, Ly6G+; A) and monocytes (CD11b+, Ly6Chigh; B) and represent 1 experiment of 3 independent experiments with similar results.
- Figure 4.
TREM-1 deletion alters activation of Kupffer cells in response to DEN and decreases expression of genes involved in inflammatory responses, cell-cycle regulation and apoptosis. A, WT and Trem1−/− male mice (n = 10 mice per group) were treated for 4 hours with DEN. Cells from total livers, isolated hepatocytes, or Kupffer cells were subjected to RNA isolation and microarray analysis. The average fold induction/decrease was obtained by comparison of Trem1−/− mice with WT mice. Fold changes in proinflammatory cytokine and chemokine gene expression (A), in genes related to NF-κB signaling pathway (B), and in genes related to JNK/p38 signaling pathway (C) are shown. Shown is one representative experiment from 2 independent experiments. Values are mean ± SD. D–F, WT and Trem1−/− mice were treated for 4 hours with DEN or left untreated (n = 4 mice per group). RNA samples extracted from livers were subjected to NanoString analysis. Decreased expression in genes involved in inflammation (D), cell-cycle regulation (E), and apoptosis (F) was observed in Trem1−/− mice post-DEN treatment. The results are expressed as mean ± SE. Statistical significance is indicated.
- Figure 5.
Adoptively transferred Kupffer cells from WT mice increased DEN-induced liver injury in Trem1−/− mice. A, immunofluorescence staining of liver sections with FITC-conjugated anti-F4/80 mAb after 48-hour injection of clodronate-containing liposomes. D, reconstitution of predepleted livers with Kupffer cells from WT mice to indicated mice. Adoptively transferred cells were observed at 18 hours by immunofluorescence staining; bar, 20 μm. Mice were given a dose of DEN at 18 hours after Kupffer cell reconstitution and ALT (B) and IL-6 (C) in serum were measured at indicated time. Data shown are means ± SD of 4 mice per group and representative of 2 independent experiments. E and F, 4 hours after DEN treatment gene expression profile of reconstituted liver was carried out using NanoString Technology. Values indicate mean ± SE of 4 mice per time point in each group. ns, not statistically significant.
- Figure 6.
TREM-1 binds HMGB1. A, HMGB1 and HSP70 present in the production of necrotic hepatocytes (PNH). PNH were isolated from DEN-treated and nontreated WT mice and subjected to Western blot analysis with indicated antibodies. Actin was used as a loading control. B, HMGB1 and not HSP70 associated with TREM-1. Necrotic hepatocytes isolated from DEN-treated WT mice were subjected to immunoprecipitation with murine recombinant TREM-1-Fc fusion protein followed by immunoblotting with indicated antibodies. C, purified murine HMGB1 and TREM-1 proteins were used as cross-linked proteins. Cross-linking reactions with bis(sulfosuccinimidyl) suberate (BS3, left) or dimethyl adipimidate (DMA, right). One of 4 similar experiments is shown. D, SPR analysis of TREM-1 and RAGE binding to immobilized HMGB1. The concentrations of RAGE (0, black; 30, yellow; 60, gray; 120, blue; and 240 nmol/L, red (left); the identical concentrations of TREM-1 and CsrA were flowed over immobilized HMGB1 on the sensor chip. Data are presented as response units (RU) over time (seconds). Data shown are representative of 3 experiments.
Additional Files
Supplementary Data
Files in this Data Supplement:
- Supplementary Methods - PDF file - 102K
- Supplementary Figure 1 - PDF file - 2.9MB, Targeting strategy for the Trem1 genomic locus and generation of Trem1-deficient mice
- Supplementary Figure 2 - PDF file - 835K, TREM-1 expression on KC of WT mice increased after DEN administration
- Supplementary Figure 3 - PDF file - 2.5MB, Deletion of TREM-1 down regulates genes associated with DNA repair and genes involved in hepatocellular carcinoma development
- Supplementary Figure 4 - PDF file - 680K, Deletion of TREM-1 inhibits cytokine and chemokine production by bone marrow-derived macrophages stimulated with the product of necrotic hepatocytes (PNH)
- Supplementary Figure 5 - PDF file - 7.2MB, The expression of TREM-1 in human liver KC
- Supplementary Figure Legends 1-5 - PDF file - 19K
Volume 72, Issue 16
